Compositions and Methods for Treating Patients Suffering from Glioma or Leukemia

ABSTRACT

Pharmaceutical compositions, kits and methods for treating tumors such as glioma and cancers such as leukemia with (R)-2-hydroxyglutarate (R-2HG) are provided, along with therapeutic regimens including treatment of a patient suffering from glioma or leukemia with a MYC-signaling inhibitor followed by or cotemporaneous with treatment with R-2HG, and optionally other chemotherapeutic agents.

PRIORITY

This application claims priority to U.S. provisional application Ser.No. 62/416,348, the entire disclosure of which is incorporated herein byreference.

GOVERNMENT INTERESTS

This invention was made with government support under contract nos. RO1CA 178454 and RO1 CA 182528 awarded by the National Institute of Health.The government has certain rights in the invention.

BACKGROUND

Many leukemia cancers, including acute myeloid leukemia (AML), a commonhematological cancer of myeloid lineage cells, generally exhibit poorprognosis in the clinic, and new treatment options are in constantdemand. Likewise, malignant gliomas, the most frequently observedprimary brain tumors, are characterized by a dismal prognosis.Interestingly, recurrent somatic mutations of the genes that code forisocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have recently beenidentified in both glioma^(1,2) and AML^(3,4). IDH1 and IDH2 are enzymesthat catalyze the oxidative decarboxylation of isocitrate to α-KG in anNAD⁺-dependent manner during the Krebs cycle. All known lesions involvearginine (R), in codon 132 for IDH1 (IDH1^(R132H)), 140 and 172 for IDH2(IDH2^(R140) and IDH2^(R172))^(5,6). In addition to losing their normalcatalytic activity, IDH mutations acquire a neomorphic enzymaticfunction that catalyzes the conversion from α-KG to the R enantiomer of2-hydroxyglutarate (R-2HG), leading to accumulation of up to millimolaramounts of R-2HG in mutant glioma and leukemia patients^(7,8).

R-2HG is structurally similar to α-KG and competitively inhibits aseries of Fe (II)/α-KG-dependent dioxygeneases⁹. Accordingly, R-2HG isconsidered as an “oncometabolite” via impairing DNA and histoneepigenetic modification and hypoxic regulation to block celldifferentiation and promote tumor transformation⁹⁻¹³. Nonetheless,several recent studies reported that inhibition of mutant IDH1(IDHi) didnot display significant effect on cell proliferation, migration, DNA andhistone methylation; instead, IDHi induced a slight increase in cancercell proliferation¹⁴⁻¹⁶.

IDH mutations occur in >70% of patients with lower-grade (II-III) braintumors and 10%-20% of AML patients with overproduction ofR-2HG^(2,17,18). Glioma patients with IDH lesions tend to have a betteroverall survival than those without^(1,2,19), and a similar trend wasreported in AML patients, although with some ensuingcontroversy^(20-22.) While it was reported that mutant IDH1 and itsproduct R-2HG induce cytokine-independent growth and blockerythropoietin (EPO)-mediated differentiation in TF-1 cells, a highlyunusual erythroleukemia cell line as it is GM-CSF-dependent¹³, theeffects of R-2HG or mutant IDH are largely undefined in leukemia cellswhose growth is cytokine-independent.

Given the apparently inconsistent data and interpretations of the roleof mutant IDH and R-2HG in the onset and prognosis of these deadlycancers, and given the crucial need for understanding these mechanisms,additional exegesis of the meaning and effects, and developing ordiscouraging therapeutics based on these understandings are an urgentand heretofore unmet need in the art.

SUMMARY

Accordingly, the results of the studies disclosed herein revealunexpected and broad anti-tumor activity of R-2HG in both leukemia andglioma involving previously unrecognized FTO/m⁶A/MYC signaling,providing a strong indication for the therapeutic potential of R-2HG.Thus, embodiments of the invention provide novel therapeuticcompositions and methods for the treatment of patients suffering fromcancers, for example leukemia and glioma.

One Embodiment of the invention provides methods for treating a tumor ina subject in need thereof comprising administering to the subject aneffective amount of R-2-hydroxyglutarate (R-2HG). In particular,patients suffering from brain tumors such as primary brain tumors(glioma) are benefited by the instant methods.

Another embodiment is directed to methods for treating cancers, such asleukemia, comprising administering to the subject an effective amount ofR-2HG. In some embodiments the patient may be pre-treated with one ormore inhibitors of MYC signaling. In other embodiments the patient maybe treated in conjunction with one or more inhibitors of MYC signalingand/or one or more chemotherapeutic agents effective for the treatmentof the cancer.

Yet another embodiment is directed to pharmaceutical compositionscomprising R-2HG and at least one pharmaceutically-acceptable carrier orexcipient. The compositions may further comprise one or more agents thatinhibi MYC signaling, and/or one or more chemotherapeutic agentseffective for treating the target cancer.

Another embodiment is directed to kits assembled for convenienttreatment of a patient suffering from a glioma or leukemia, the kitscomprising a first vial comprising R-2HG, and at least one second vialcomprising an agent effective for inhibiting MYC signaling.

The unexpected intrinsic and broad anti-tumor activity of R-2HG is shownto implicate suppression of FTO/m⁶A/MYC signaling. In addition, R-2HGactivity in sensitizing cancer cells to the treatment of MYCinhibitor(s) and other therapeutic agents is demonstrated. These studiesnot only reveal the unrecognized activities of R-2HG and the functionalimportance of FTO, MYC, and RNA epigenetics (herein m⁶A RNAmodification) in R-2HG-associated pathways, which provide novel insightsinto the molecular mechanisms underlying tumor pathogenesis and drugresponse, but also provide novel therapeutic strategies to treat cancerswith or without IDH mutations.

These and other embodiments and aspects will be further detailed andclarified by reference to the Drawings and to the Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor.

Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1A-FIG. 1K evidence the anti-leukemic activity of R-2HG. FIG. 1A)Relative cell viabilities of 27 human leukemia cell lines treated with300uM cell-permeable R-2HG for 24, 48, 72 and 96 h. The varying colorrepresents different time points; the diameter of the specific indicatesthe relative cell viability. FIG. 1B) Relative cell viabilities of the27 cell lines treated with different concentrations of R-2HG at 96 h.The color represents R-2HG concentration; the diameter representsrelative cell viability. FIG. 1C) Effects of R-2HG on cell cycle FIG. 1Dand cell apoptosis FIG. 1E) in sensitive (NOMO-1) cells. FIG. 1F)Effects of R-2HG on colony-forming capacity and FIG. 1G) cell viabilityof leukemic blast cells isolated from primary AML patients. FIG. 1H)Schematic illustration of the ex vivo strategy. FIG. 11) Kaplan-Meiercurves showing the leukemic progression of R-2HG- or PBS-treatedsensitive cells in vivo. FIG. 1J) Spleen and liver weights from leukemicNSGS mice injected with R-2HG- or PBS-treated NOMO-1 cells. FIG. 1K)Engraftments of PBS- or R-2HG-treated NOMO-1 cells in PB (peripheralblood), BM (bone marrow) and spleen of leukemic NSGS recipient mice. ForKaplan-Meier curve, P values were calculated by log-rank test.

FIG. 2A-FIG. 2D identify genes and pathways related to R-2HG response.FIG. 2A) Identification of potential α-KG-dependent dioxygenases andsignaling pathways responsible for varying sensitivities to R-2HGtreatment. Upper panel: the top 10 α-KG-dependent dioxygenases showingpositive correlation with R-2HG sensitivity; Lower panel: the top 3signaling pathways distinguishing sensitive and resistant leukemiacells. FIG. 2B) Global effects of R-2HG treatment in sensitive leukemiacells. Upper panel: the top 10 α-KG-dependent dioxygenasesdown-regulated by R-2HG; Lower panel, the top 3 signaling pathwayssuppressed by R-2HG in sensitive leukemia cells. FIG. 2C) Gene setenrichment analysis (GSEA)²⁸ of significantly differentially expressedgenes in four groups of comparisons, seven pathways are enriched in allthe comparisons. FIG. 2D) the normalized enrichment scores of MYC, G2Mand E2F signaling pathways in the four groups of comparisons.

FIG. 3A-FIG. 3J set forth data demonstrating that R-2HG induces m⁶Amodification via inhibiting FTO. FIG. 3A) R-2HG treatment increasesglobal m⁶A levels in sensitive leukemia cells. MB, methyl blue,represents the RNA loading control. FIG. 3B) R-2HG has little effects onm⁶A RNA modification in resistant leukemic cells. FIG. 3C) Schematicillustration of Drug Affinity Responsive Targets Stability (DARTS). FIG.3D) DARTS assays indicate R-2HG binds direct to FTO, but not ALKBHS.FIG. 3E) Effects of R-2HG on FTO and ALKBHS expression. FIG. 3F) leftpanel—effects of FTO overexpression or knockdown (by shRNA) on cellproliferation/growth, right panel—cell viability, FIG. 3G) as well as onthe global m⁶A modification in MA9.3ITD cells. FIG. 3H) Effects of FTOoverexpression or knockdown on cell growth/viability and FIG. 3I) m⁶Amodification in U937 cells. FIG. 3J) left panel—knockdown of FTOdecreases sensitivity to R-2HG treatment in sensitive (NOMO-1) cells,right panel—forced expression of FTO increased sensitivity to R-2HG inresistant (K562) leukemia cells. *, P<0.05; **, P<0.01; ***, P<0.001;t-test.

FIG. 4A-FIG. 4K provide evidence that R-2HG and FTO regulate MYCexpression via manipulating m⁶A modification. FIG. 4A) Schematicillustration of m⁶A-sequencing (m⁶A-seq) with PBS- or R-2HG-treatedsensitive cells (NOMO-1 as a representative). FIG. 4B) The density(line) and frequency (histogram) distributions of m⁶A peaks in NOMO-1cells with R-2HG versus PBS-treatment. Fold enrichment indicates thepeaks are more enriched in the R-2HG treated sample than in thePBS-treated sample. FIG. 4C) Summary of changed m⁶A peaks after R-2HGtreatment. Volcano plot representation of differentially methylatedpeaks in sensitive cell line with R-2HG treatment versus PBS-treatment.The significantly increased (red) or decreased (blue) m⁶A peaks (P<0.01)are highlighted. FIG. 4D) GSEA analysis of genes with a significantincrease in m⁶A modification on transcripts after R-2HG treatment. FIG.4E) The m⁶A abundance in MYC mRNA in R-2HG- or PBS-treated NOMO-1 cells.FIG. 4F) Gene-specific m⁶A qPCR validation of m⁶A level changes of MYCin NOMO-1 cells. FIG. 4G) Luciferase and mutagenesis assays. 293T cellswere co-transfected with MYC-5′UTR (left panel) or MYC-CDS (right panel)bearing wild-type or mutant (m⁶A replaced by T) m⁶A motifs, togetherwith wild-type FTO, FTO mutant or control vector. FIG. 4H) Effects ofR-2HG on MYC mRNA stability in sensitive (NOMO-1) or resistant (K562)leukemia cells. FIG. 4I) Effects of knockdown m⁶A reader YTHDF2 on MYCmRNA stability. FIG. 4J) Profiles of m⁶A peaks on MYC transcripts inPBS- or R-2HG-treated sensitive (MA9.3ITD) or resistant (MA9.3RAS) cellswith or without FTO knockdown (for MA9.3ITD) or FTO overexpression (forMA9.3RAS), as detected by m⁶A-seq. FIG. 4K) Model of anti-leukemicfunction of R-2HG through FTO inhibition. *, P<0.05; **, P<0.01; ***,P<0.001; t-test. Error bar, mean±SD.

FIG. 5A-FIG. 5K demonstrate that the abundance of FTO and MYC controlssensitivity of leukemic cells to R-2HG. FIG. 5A) Mutant IDH(IDH1^(R132H)) also inhibits FTO and MYC expression in sensitive cells(NOMO-1 and U937), FIG. 5B) but not in resistant cells (NB4). FIG. 5C)Effects of IDH1^(R132H) on global m⁶A levels in the above sensitive andresistant leukemia cells. FIG. 5D) Function of IDH1^(R132H) on cellcycle, FIG. 5E) proliferation, and FIG. 5F) apoptosis in the abovesensitive and resistant cells. FIG. 5G) Venn diagram shows the sharedsignaling pathways (or gene sets) of the 4 indicated groups ofcomparisons. The sensitive, resistant and healthy control samples arethe R-2HG-sensitive or -resistant leukemic cell lines and normal controlsamples shown in FIG. 2A. The other samples listed in the plot are humanprimary AML samples of the TCGA dataset⁴⁰: IDH mutant, the AML sampleswith mutations in IDH1 and/or IDH2; IDH WT, the AML samples withwild-type IDH genes; IDH WT (NK), the normal-karyotype AML samples withwild-type IDH genes. FIG. 5H) Relative expression levels of FTO, ALKBH5,MYC, CDK4 and CDK6 (the latter two are critical targets of MYC) inprimary AML patients with or without IDH mutation as well as in healthycontrols. FIG. 5I) Expression pattern of FTO and MYC in sensitive andresistant cells with or without R-2HG treatment. FIG. 5J) MYCoverexpression renders sensitive leukemic cells resistant to R-2HG. FIG.5K) Suppression of the hyper-activated MYC signaling by JQ1 sensitizesR-2HG-resistant leukemic cells to R-2HG or IDH mutant.

FIGS. 6A-6AA demonstrate the effects of R-2HG on theproliferation/growth of leukemic cells. Individual 27 leukemic celllines were seeded in 96-well plate at 10,000 cells/well, under thetreatment with 20 uM, 100 uM, or 300 uM cell-permeable R-2HG, or withvehicle control (PBS), with FIG. 6A) depicting NOMO-1, FIG. 6B) U937,FIG. 6C) MA9.3ITD, FIG. 6D) ML-2, FIG. 6E) MONOMAC-6, FIG. 6F) MA9.3,FIG. 6G) THP1, FIG. 6H) MA9.6, FIG. 6I) PL21, FIG. 6J) MA9.6ITD, FIG.6K) KOPN-1, FIG. 6L) SKNO-1, FIG. 6M) MA9.6RAS, FIG. 6N) MV4-11, FIG.60) ME-1, FIG. 6P) KASUMI-1, FIG. 6Q) KOCL69, FIG. 6R) JURKAT, FIG. 6S)KOCL481, FIG. 6T) KOCL50, FIG. 6U) KOCL45, FIG. 6V) MA9.3RAS, FIG. 6W)TF-1, FIG. 6X) HEL, FIG. 6Y) KOCL51, FIG. 6Z) K562, and FIG. 6AA) NB4.The cell proliferation was detected by MTT assay via recordingabsorbance at 570 nm at indicated time points (i.e., 0, 24, 48, 72 and96 hours post seeding). Detailed information about the 27 leukemia celllines, including fusion genes, karyotype and FAB subtypes, is set forthin Table 1. *, P<0.05; **, P<0.01; ***, P<0.001; t-test. depicts cellproliferation of the NOMO-1 cell line.

FIGS. 7A-7AA show the effects of R-2HG treatment on cell viability ofthe 27 leukemia cell lines. Relative cell viabilities of the leukemiccells treated with varying concentrations of R-2HG at varying timepoints are shown. *, P<0.05; **, P<0.01; ***, P<0.001; t-test.Specifically, FIG. 7A) depicts the cell viability of NOMO-1, FIG. 7B)U937, FIG. 7C) MA9.3ITD, FIG. 7D) ML-2, FIG. 7E) MONOMAC-6, FIG. 7F)MA9.3, FIG. 7G) THP1, FIG. 7H) MA9.6, FIG. 7I) PL21, FIG. 7J) MA9.6ITD,FIG. 7K) KOPN-1, FIG. 7L) SKNO-1, FIG. 7M) MA9.6RAS, FIG. 7N) MV4-11,FIG. 70) ME-1, FIG. 7P) KASUMI-1, FIG. 7Q) KOCL69, FIG. 7R) JURKAT, FIG.7S) KOCL481, FIG. 7T) KOCL50, FIG. 7U) KOCL45, FIG. 7V) MA9.3RAS, FIG.7W) TF-1, FIG. 7X) HEL, FIG. 7Y) KOCL51, FIG. 7Z) K562, and FIG. 7AA)NB4.

FIG. 8A-FIG. 8C demonstrate the effects of R-2HG on cell cycle andapoptosis. FIG. 8A) Left, flow cytometry analyzing the cell cycle inNOMO-1 (upper panel), U937 (middle panel) and NB4 (lower panel) leukemiccells, stained with Propidium iodide (PI). Right, the summaries of cellcycle distributions of the leukemic cells with or without R-2HGtreatment, based on three independent experiments. FIG. 8B) Left, flowcytometry of NOMO-1 (upper panel), U937 (middle panel) and NB4 (lowerpanel) leukemia cells, stained with Hoechst 33342 and Pyronin Y. Numbersin quadrant represented the percent cells at G0 (black), G1 (blue) andS/G2/M (red) stages respectively. Right, the summarized results based onthree independent experiments. FIG. 8C) Flow cytometry analysis ofapoptosis in NOMO-1, U937 and NB4 cells, stained with FITC-labeledAnnexin V and PI, along with the summarized results based on threeindependent experiments. *, P<0.05; **, P<0.01; ***, P<0.001; t-test.

FIG. 9A-FIG. 9D show the effects of R-2HG on cell viability andproliferation/growth of two GM-CSF-dependent leukemia cell lines, TF-1and SKNO-1, under cytokine-normal or -poor conditions. FIG. 9A) Effectsof R-2HG on cell proliferation (upper panel; cell density detected byMTT assays), viability (middle panel; detected by MTT assays) and growth(lower level; detected by cell number counts) of TF-1 cells culturedwith normal GM-CSF (2 ng/mL) or FIG. 9B) GM-CSF-poor condition (0.1ng/mL). FIG. 9C) Effects of R-2HG on cell proliferation (upper panel),viability (middle panel) and growth (lower level) of SKNO-1 cellscultured with normal GM-CSF (10 ng/mL) or FIG. 9D) GM-CSF-poor condition(0.1 ng/mL). *, P<0.05; **, P<0.01; t-test.

FIG. 10A-FIG. 10D set forth the effects of R-2HG on the engraftment andleukemic blast cell proportion of R-2HG-sensitive leukemic cells in NSGSrecipient mice. FIG. 10A) FACS analysis of CD45⁺ cells in peripheralblood (PB), bone marrow (BM) and spleen of three representative leukemicNSGS recipient mice xeno-transplanted with PBS-treated, or FIG. 10B)R-2HG-treated NOMO-1 cells. Percentage of human CD45⁺ cells representedthe engraftment of human leukemic cells into NSGS mice. FIG. 10C) FACSanalysis of c-Kit⁺ Mac-1⁺ cell populations in BM and spleen of threerepresentative leukemic NSGS recipient mice xeno-transplanted withPBS-treated, or FIG. 10D) R-2HG-treated NOMO-1 cells.

FIG. 11A-FIG. 11K set forth data re the α-KG dependent dioxygenasesshowing positive correlation with R-2HG sensitivity as detected byRNA-seq, followed by qPCR validation. FIG. 11A) The α-KG dependentdioxygenases that exhibited a positive correlation (r≥0.2) in expressionwith sensitivity of the leukemic cells to R-2HG treatment as detected byRNA-seq (see FIG. 2A). The top 10 α-KG dependent enzymes that show themost significantly positive correlation are highlighted and qPCRanalysis of expression patterns of the aforementioned top 10 α-KGdependent enzymes in 30 human leukemic samples (including the 27 celllines shown in FIG. 1A and FIG. 1B and 3 primary leukemic samples) andin an expanded cohort of healthy control samples, including 16 normalmononuclear cell (MNC) samples isolated from peripheral blood or bonemarrow of healthy donors, and 10 CD34⁺ and 12 CD34⁻ MNC samples isolatedfrom cord blood samples, along with Pearson correlation analysis of thecorrelation between expression levels of the individual α-KG dependentenzymes and sensitivity of the leukemic samples to R-2HG treatmentacross the 27 leukemic cell lines. The relative expression levelsamongst different normal control sample groups and the leukemic samplegroup (upper panels) and Person correlative analysis (lower panels) ofFIG. 11B) P4HB, FIG. 11C) LCP1, FIG. 11D) PHF2, FIG. 11E) PKM, FIG. 11F)PHF8, FIG. 11G) PLODS, FIG. 11H) KDM2B, FIG. 111) PHYH, FIG. 11J) FTO,or FIG. 11K) ALKBH7 are shown. *, P<0.05; **, P<0.01; t-test.

FIG. 12A-FIG. 12C set forth the genes that are significantlydown-regulated or up-regulated by R-2HG in NOMO-1 and Ma9.3ITD cells,and expression patterns of the α-KG-dependent enzymes in these two celllines with or without R-2HG treatment, as detected by RNA-seq. FIG. 12A)Summary of the RNA-seq results. Volcano plot representation ofdifferential expression genes in NOMO-1 (left) and MA9.3ITD (right) celllines with R-2HG treatment versus PBS-treatment. The red and green dotsrepresent the genes with a significant (P<0.05, log_(1.5) (foldchange)>1) increase and decrease, respectively, in expression upon R-2HGtreatment. FIG. 12B) The significantly dys-regulated genes in bothNOMO-1 and MA9.3ITD cell lines upon R-2HG treatment. Red and green dotsrepresent the genes significantly increased and decreased, respectively,in both cell lines (log_(1.5)(fold change)>1). FIG. 12C) The expressionchanges of all the α-KG dependent/related dioxygenases (with expressionvalues in all the four samples) after 300 uM R-2HG treated for 48 hoursin NOMO-1 and MA9.3ITD cells.

FIG. 13A-FIG. 13G show the core signaling pathways identified byRNA-seq. Based on the RNA-seq data from the samples shown in FIG. 2A(i.e., four R-2HG-sensitive leukemic samples, five R-2HG-resistantleukemic samples, and four healthy control samples) and the RNA-seq datafrom the samples shown in FIG. 2B (i.e., NOMO-1 and MA9.3ITD leukemicsamples treated with PBS or R-2HG), GSEA identified 7 core enriched genesets (or signaling pathways) from the following four groups ofcomparisons: sensitive leukemia cells vs. resistant leukemia cells;sensitive leukemia cells vs. healthy control cells; R-2HG-treated NOMO-1vs. PBS-treated NOMO-1; and R-2HG-treated MA9.3ITD vs. PBS-treatedMA9.3ITD. Among the 7 gene sets, FIG. 13A) MYC targets V1, FIG. 13B) MYCtargets V2, FIG. 13C) G2M checkpoint and FIG. 13D) E2F targets wereconsistently enriched in resistant cells compared with sensitive cells,and also enriched in sensitive cells compared with healthy controls, andnotably suppressed by R-2HG treatment in both NOMO-1 and MA9.3ITD cells,whereas the other three genes sets including FIG. 13E) Cholesterolhomeostasis, FIG. 13F) Inflammatory response and FIG. 13G) TNFAsignaling via NF-kB show the largely opposite patterns. ES, enrichmentscore. P<0.001 and FDR<0.05 were used as cut-off for statisticsignificance.

FIG. 14A-FIG. 14F show the core genes from enriched MYC, G2M and E2Fsignaling pathways. FIG. 14A) a Venn diagram displaying the coreenrichment genes amongst the four gene sets including ‘MYC targets V1’,‘MYC targets V2’, ‘G2M checkpoint’ and ‘E2F targets’ shared by both‘sensitive vs. resistant’ and ‘sensitive vs. healthy control’comparisons. FIG. 14B) Heat map of the 146 shared core enrichment genes.They showed the highest abundance in R-2HG-resistant leukemia cells andthe lowest abundance in healthy controls, with a middle level ofabundance in R-2HG-sensitive leukemic cells. FIG. 14C) Venn diagramshowing the core enrichment genes amongst the aforementioned four genesets shared by both ‘R-2HG-treated NOMO-1 vs. PBS-treated NOMO-1’ and‘R-2HG-treated MA9.3ITD vs. PBS-treated MA9.3ITD’ comparisons. FIG. 14D)Heat map of the 185 shared core enrichment genes, which wereconsistently and significantly suppressed by R-2HG in both NOMO-1 andMA9.3ITD cells. FIG. 14E) Relative expression of major component genes(including CCNA2, CDK2, CDK4, CDK6, DDX21, MCM2 and MCMI) of the MYCpathways in sensitive (NOMO-1) cells or FIG.

14F) resistant (K562) cells with or without R-2HG treatment, as detectedby qPCR. *, P<0.05; **, P<0.001; t-test.

FIG. 15A-FIG. 15H set forth a correlation analysis betweenDNA/RNA/histone demethylases expression and sensitivity to R-2HG, aswell as the effects of R-2HG on

DNA and histone methylation. FIG. 15A) Schematic illustration of enzymesmediating demethylation of DNA, RNA and protein. All these enzymes arethe Fe (II)/α-KG-dependent dioxygenase. TET family members (TET1/2/3)mediate demethylation of DNA through converting 5-methylcytosine (5 mC)to 5-hydroxymethylcytosine (5 hmC). ALKBHS and FTO, belonging to AlkBfamily, are responsible for the removal of m⁶A modification on RNA. KDM(histone lysine demethylase) and JHMD (Jumonji Domain-containing histonedemethylase) catalyzing the demethylation of histone. FIG. 15B) Relativeexpression of the DNA demethylase genes (TET1/2/3), FIG. 15C) RNAdemethylase genes ALKBH5 and FTO, and FIG. 15D) histone demthylase genesKDM2A, KDM4A and JMJD6 in leukemia cells and healthy controls (upperpanels), and Pearson correlation analysis between their expression andsensitivities to R-2HG in the leukemia cells (lower panels). FIG. 15E)The 5 hmC levels in R-2HG-sensitive leukemic cells, or FIG. 15F)R-2HG-resistant leukemic cells with or without R-2HG treatment for 48hours. 5 hmC levels were determined by dot blot. MB, methylene blue asthe loading control. FIG. 15G) The histone methylation status afterR-2HG treatment in sensitive leukemic cells, or FIG. 15H) resistantleukemic cells. β-actin was used as loading control. *, P<0.05; **,P<0.01; t-test.

FIG. 16A-FIG. 16I are a transcriptome-wide m⁶A-seq and analysis of m⁶Apeaks. FIG. 16A) Identification of m⁶A peaks by two algorithms. Thelayers from outer to inner represents the m⁶A peaks identified by MACS2,exomePeak, and by both algorithms, respectively. FIG. 16B) The densitydistribution of m⁶A peaks across the length of mRNA transcripts. Eachregion of 5′untranslated region (5′ UTR), coding region (CDS), and3′untranslated region (3′ UTR) was split into 100 segments, and thepercentage of m⁶A peaks that fall within each segment was determined.FIG. 16C) The proportion (upper panel) and enrichment (lower panel) ofthe m⁶A peak distribution in the 5′UTR, start codon, CDS, stop codon or3′UTR region across the mRNA transcripts. The enrichment was calculatedby the number of m⁶A peaks normalized by the length of the region. FIG.16D) Distribution of the m⁶A peaks in exonic or intronic regions ofprotein-coding genes or non-coding genes, or in other regions. FIG. 16E)The m⁶A motifs detected by HOMER using a predominant consensus motifDRACH ([G/A/U] [G/A] m⁶AC [U/A/C]). FIG. 16F) Distribution of theincreased m⁶A peaks in RNA regions. FIG. 16G) The pie shows thepercentage of nucleotides mapped to the increased or decreased m⁶A peaksin 5′UTR, start codon, CDS, stop codon, 3′UTR, intron and non-coding RNA(ncRNA). FIG. 16H) Distribution of genes with a significant change inboth m⁶A level (P<0.01) and RNA expression level in NOMO-1 cells afterR-2HG treatment. FIG. 16I) The CLIP-seq data (GSE49339) indicates thepredominant binding of YTHDF2 on MYC mRNA.

FIG. 17A-FIG. 17D show the effects of R-2HG, FTO and m⁶A “reader” YTHDF2on

MYC expression. FIG. 17A) Effects of R-2HG on FTO and MYC expression insensitive and resistant leukemia cell. R-2HG notably down-regulated FTOand MYC expression at mRNA levels in NOMO-1 (left panel) and MA9.3ITD(middle panel) sensitive leukimia cells, while it had little effects onFTO and MYC expression in K562 (right panel) resistant cells. FIG. 17B)Inhibition of FTO and MYC expression at protein levels by R-2HG insensitive (NOMO-1 and MA9.3ITD) cells but not in resistant (K562) cells.FIG. 17C) Effect of FTO on MYC expression. Forced expression ofwild-type FTO increased MYC expression compared with mutant FTO orcontrol group, and FTO knockdown decreased MYC expression, in sensitive(MA9.3ITD) leukemia cells. FIG. 17D) Knockdown of YTHDF2 expressioncaused a significant increase in MYC expression level. **, P<0.01;t-test.

FIG. 18A-FIG. 18F identify the signaling pathways determiningsensitivity to R-2HG and the synergistic action between R-2HG andchemotherapy drugs. FIG. 18A) The 5 core gene sets shared by four groupscomparisons, including ‘IDH mutant vs. IDH WT’ (i.e., AML patients withIDH mutations vs. AML patients without IDH mutations), FIG. 18B) ‘IDHmutant vs. IDH WT (NK)’ (i.e., AML patients with IDH mutations vs. AMLpatients with normal karyotype and without IDH mutations), FIG. 18C)‘sensitive vs. resistant’ (i.e., R-2HG-sensitive leukemic cell lines vs.R-2HG-resistant leukemic cell lines; see the samples in FIG. 2A), andFIG. 18D) ‘sensitive vs. healthy control’ (i.e., R-2HG-sensitiveleukemic cell lines vs. healthy control samples; see the samples in FIG.2A). FIG. 18E) The IC50 values of JQ-1 in AML patients with IDHmutations or wild-type IDH genes. FIG. 18F) The synergistic effectsbetween R-2HG and clinical chemotherapeutic drugs, including ATRA,Daunorubicin, AZA and Decitabine, on inhibiting leukemic cell viability(using MONOMAC 6 as a representative). *, P<0.05; **, P<0.01; t-test.

FIG. 19A-FIG. 19C evidence demonstrating the anti-tumor effects of R-2HGon brain tumor cells. FIG. 19A) R-2HG suppresses cellproliferation/growth in the 8 brain tumors cells. All the cells wereplated in 96-well plate at 5,000-10,000 cells/well and the cellproliferation was assessed by MTT assays. FIG. 19B) R-2HG inhibits cellviability of glioma cells in a dose-dependent manner. The relative cellviability of the 8 glioma cell lines were detected by MTT 96 hourspost-treatment with 20 μM, 100 μM or 300 μM R-2HG. FIG. 19C) R-2HGdecreases cell viability of glioma cells in a time-dependent manner. Allthe brain tumor cells were treated with 300 μM R-2HG for 24, 48, 72 or96 hours. *, P<0.05; **, P<0.01; t-test.

DETAILED DESCRIPTION

Prior to the investigations set forth herein, R-2-hydroxyglutarate(R-2HG), which is accumulated in subjects exhibiting IsocitrateDehydrogenase 1 and 2 (IDH1 and IDH2) mutations, was widely consideredto be an oncometabolite via interfering with α-ketoglutarate(α-KG)-dependent dioxygenases. However, these investigations reveal thatR-2HG actually exerts a broad anti-leukemic activity in vitro and invivo by inhibiting leukemia cell proliferation/viability and promotingcell-cycle arrest and apoptosis. Mechanistically, in R-2HG-sensitivecells, R-2HG dramatically induces global N⁶-methyladenosine (m⁶A) RNAmodification, mainly through suppression of the expression and activityof FTO, an RNA demethylase. Consequently, the increased m⁶A modificationcauses less stability of MYC transcripts, leading to the suppression ofMYC-associated signaling pathways. Mutant IDH recapitulates the effectof R-2HG. Interestingly, while high abundance of FTO sensitizes leukemiccells to R-2HG, hyperactivation of MYC signaling confers resistance,which can be reversed by pharmaceutical inhibition of MYC signaling.R-2HG also shows synergistic anti-tumor effects with other therapeuticagents in leukemia and glioma. Thus, the data highlights the therapeuticpotential and efficacy of treatment of cancer with R-2HG.

Notably, R-2HG predominantly increases global m⁶A RNA modification,rather than histone or DNA methylation, in sensitive leukemia cells, asdetected by both m⁶A dot blot and transcriptome-wide m⁶A-seq assays. Thesensitivity of leukemic cells to R-2HG is positively correlated with theexpression level of FTO, an Fe(II)/α-KG dependent m⁶Ademethylase^(33,41), and R-2HG binds directly to FTO (suppressing itsenzymatic activity) and strikingly, R-2HG treatment also causes thedown-regulation of FTO expression through unrecognized mechanism(s).Experiments set forth herein show that FTO plays an oncogenic role inleukemia and its knockdown mimics effect of R-2HG. Together, our datasuggest that FTO is a direct target of R-2HG and itsfunctional/expressional suppression caused by R-2HG is likelyresponsible for R-2HG-mediated anti-leukemic effect and increase ofglobal m⁶A modification. Further, the high abundance of FTO expressionis a feature of R-2HG-sensitive leukemic cells, and manipulatingexpression level of FTO can change the sensitivity/resistance ofleukemic cells to R-2HG.

A few leukemic cell lines are resistant to R-2HG treatment. RNA-seqprofiling assays indicate that R-2HG-resistant leukemic cells have ahyper-activation of the MYC, G2M and E2F signaling pathways, relative toR-2HG-sensitive leukemic cells, while the latter has a relatively higheractivation of these pathways than healthy controls. Interestingly, thesesignaling pathways are also highly activated in AML patients carryingIDH mutations. Moreover, forced expression of MYC, a master transcriptregulator and universal transcriptional amplifier that regulates allthese pathways^(38,39), renders R-2HG-sensitive leukemic cells resistantto R-2HG. Conversely, JQ1 inhibition of MYC signaling confersR-2HG-sensitivty in R-2HG-resistant leukemic cells. Thus, the R-2HGresistance in leukemic cells may be attributed to the hyper-activationof MYC signaling (and the associated pathways).

Intriguingly, the MYC, G2M and E2F signaling pathways are also the mostresponsive pathways suppressed by R-2HG in sensitive leukemic cells.Examples set forth herein show that R-2HG treatment causes a substantialincrease in the abundance of m⁶A modification on MYC mRNA transcripts,especially at the 5′UTR and CDS regions, in R-2HG-sensitive leukemiccells (but not in R-2HG-resistant ones), associated with a substantialdown-regulation of MYC expression. Rescue assays and luciferasereporter/mutagenesis assays suggest that the effect of R-2HG on m⁶Amodification and regulation of MYC expression relies on R-2HG-mediatedsuppression of FTO activity/expression and FTO's demethylase activity,as well as the associated changes in m⁶A modification on MYCtranscripts. Moreover, R-2HG-mediated increase of m⁶A modification onMYC transcripts tends to be recognized by m⁶A “reader” YTHDF2³⁷ andeventually leads to mRNA degradation. Thus, these studies reveal a newmolecular mechanism by which R-2HG suppresses the activity/expression ofFTO, and thereby increases the m⁶A abundance on key downstream targettranscripts (e.g., MYC) and post-transcriptionally regulates theirexpression (e.g., through RNA degradation), leading to anti-tumor effect(FIG. 4K). Together, this work reveals a previously unrecognized,m⁶A-modification-associated oncogenic signaling (i.e., FTO

m⁶A modification

MYC, etc.) in leukemic cells and its suppression by R-2HG is a majormechanism responsible for R-2HG's broad anti-leukemic effect.

Collectively, the data suggest that the sensitivity/resistance ofleukemic cells to R-2HG is controlled by the concentration of FTO andMYC, and is dose-dependent. The higher level of FTO abundance is oftenassociated with the higher sensitivity of the leukemic cells to R-2HG,likely attributed to the higher functional importance of FTO and itsassociated oncogenic signaling on the survival of such leukemic cells.Although MYC and its associated signaling pathways are criticaldownstream targets of FTO and R-2HG, too high abundance of MYC likelycannot be sufficiently depressed by R-2HG (or FTO suppression) to athreshold that can trigger anti-leukemic effect. Thus, hyper-activationof MYC signaling pathway(s) renders leukemic cells resistant to R-2HG.

While exogenous mutant IDH displays a similar anti-leukemic effect toR-2HG in R-2HG-sensitive leukemic cells, it shows no inhibitory effectin resistant cells in which MYC signaling is hyper-activated.Interestingly, R-2HG treatment causes a substantial decrease in global 5hmC abundance (FIG. 16F) in R-2HG-resistant cells, which might be due toR-2HG-mediated inhibition of the activity of TET2^(13,42), awell-recognized tumor suppressor gene^(42,43). Thus, while IDH mutant'spotential anti-leukemic effect is abrogated by hyper-activated MYCsignaling, IDH mutant may also contribute to leukemic cell survival tosome degree by inhibition of TET2, and this may explain why IDHmutations still occur in 10%-20% of AML cases^(17,18) and such AMLpatients are more responsive to hypo-methylating agents⁴⁴.

Also as demonstrated herein, R-2HG decreases cell proliferation andviability in human brain tumor cells, suggesting that R-2HG may have anintrinsic anti-tumor activity in a broad array of tumors; although suchactivity can be compromised by a strong oncogenic signaling (e.g., MYCsignaling) in patients with IDH mutations. Remarkably, this datademonstrates that R-2HG exhibits a synergistic effect with JQ1 and acohort of first-line therapeutic agents (e.g., azacitidine, decitabine,ATRA, and daunorubicin) in inhibition of leukemic cell growth/survival.Consistent with these findings, previous studies show that leukemiapatients with IDH mutations tend to be more sensitive to treatment withhypomethylating agents such as azacitidine and decitabine⁴⁴, ATRA⁴⁵, orstandard chemotherapy (daunorubicin and others)^(20,21,) than thosewithout. Similarly, glioma patients carrying IDH mutations also have amore favorable overall survival than those without^(2,19), which mightalso be attributed to the possibility that endogenous R-2HG sensitizestumor cells to standard therapies (e.g., TMZ) applied to brain tumorpatients. Thus, besides its intrinsic anti-tumor activity, R-2HG likelyalso contributes to the drug response of cancer cells. Thus, thecombinations of R-2HG (exogenous one, or that induced by endogenous IDHmutations) with MYC inhibitor(s) and other widely used therapeuticagents (e.g., Azacitidine, Decitabine, ATRA, or Daunorubicin) mayrepresent more effective novel therapeutic strategies to treat leukemiaand glioma (and likely also other cancer types). It is contemplated thatdifferent subtypes of cancers may need different combinations oftreatment.

Some embodiments provide methods of treating a tumor or a cancer in asubject in need thereof comprising administering to the subject aneffective amount of R-2-hydroxyglutarate (R-2HG). According to morespecific embodiments, the subject is suffering from a brain tumor, andaccording to even more specific embodiments the brain tumor comprises aprimary brain tumor/glioma. In other specific embodiments, the cancercomprises a hematologic cancer, and even more specifically, the cancercomprises leukemia. According to very specific embodiments, the cancercomprises acute myeloid leukemia (AML). Notably, recent data generatedby the present investigators suggests that S-2HG may be equally aseffective in the methods disclosed herein and work is currently underwayto confirm with respect to both S-2-HG and racemic 2-HG.

According to one embodiment, at least one agent effective for inhibitingMYC signaling prior is administered to the patient prior toadministering the R-2HG. Exemplary such agents are set forth in Table 4.Patients exhibiting a resistant phenotype either prior to commencingtreatment or acquired epigentically after initiation of treatment may beparticularly benefited by this embodiment. For example, the patient mayexhibit a mutant form of IDH1 and/or an IDH2.

According to another embodiment, one or more chemotherapeutic agents maybe administered in conjunction with the R-2HG. Exemplarychemotherapeutic agents include but are not limited to all transretinoic acid (ATRA), azacitidine (AZA), daunorubicin, and decitabine.“In conjunction” as utilized herein is intended to mean as part of thesame therapeutic regimen and includes, for example, prior to, subsequentto, and cotemporaneous with administration of R-2HG. In specificembodiments administering comprises cotemporaneous administration. Inother specific embodiments administering comprises administering as asecondary therapeutic subsequent to tolerance. In additional specificembodiments, administering comprises administering at least one smallmolecule MYC-signaling inhibitor selected from Table 4, R-2HG, and atleast one chemotherapeutic agent in the same therapeutic regimen.

In some embodiments, R-2HG may be modified, for example to increasemembrane permeability. According to specific embodiments, the R-2HG isester modified. For purposes of the Examples set forth herein “R-2HG” isester-modified R-2HG. In very specific embodiments, the R-2HG is estermodified in accordance with the following structure:

Another embodiment is directed to a pharmaceutical compositioncomprising R-2HG (or S-2HG, or a racemic mixture thereof), and one ormore pharmaceutically-acceptable carriers and/or excipients. Accordingto more specific embodiments, the pharmaceutical composition furthercomprises at least one agent that inhibits MYC signaling. Exemplary suchagents are set forth in Table 4.

In one aspect, the R-2HG may be recombinant R-2HG. Recombinant forms ofR-2HG are known in the art, for example production of recombinant R-2HGsuitable for the instant methods and compositions is disclosed inLosman, J. A. et al. Science 2013, Mar. 29:339(6127) pp1621-5, theentire disclosure of which is incorporated herein by reference. R-2HGmay be administered as modified R-2HG, for example as ester-modifiedR-2HG. Pharmaceutical dosage forms suitable for administration includeoral and parenteral.

Embodiments of the pharmaceutical composition may be formulated for oralor parenteral administration. According to preferred embodiments, thepharmaceutical compositions are formulated for parenteraladministration, for examples as injectable suspensions. Generally,pharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In allcases, the form must be sterile and must be fluid to the extent thateasy syringability exits. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol),suitable mixtures thereof, and vegetable oils. In some embodiment, theactive may be loaded into/onto nano-carriers, including nano-carriersfunctionalized to target specific tumor or cancer cells.

R-2HG is typically stored in millimolar concentrations of between 100and 300 mM. The molecular weight of ester-modified R-2HG is 282.31g/mol. Specific embodiments of the pharmaceutical compositionsformulated as injectable suspensions comprise between about 10 and 500,50 and 400, 100, and 300, 150 and 250, or about 200 μM R-2HG by weight,and may be administered in a dose of about 1-10 mg R-2HG per kg bodyweight. However, it will be readily apparent to a person of ordinaryskill in the art that specific concentrations and doses will varyaccording to the characteristics and disease status of an individualpatient, and as with most pharmaceutical compositions formulated for thetreatment of cancer, the concentrations of active will be variable andpersonalized; yet readily determinable by the ordinary clinician.

According to other embodiments, kits for convenient clinical treatmentof a patient suffering from a glioma or leukemia are also provided. Inspecific embodiments the kit may comprise a first vial comprising R-2HG,and at least one second vial comprising an agent effective forinhibiting MYC signaling. According to specific embodiments a kit ispackaged as relevant to a treatment time frame, and comprises more thanone sets of first and second vials. According to very specificembodiments, the at least one second vial comprises an agent selectedfrom the group consisting of the agents set forth in Table 4.

The experiments set forth herein demonstrate that contrary to previouslywidely-embraced beliefs otherwise, R-2HG actually exhibits a broadanti-leukemia function in the vast majority cases of a variety of humanleukemia cell lines, as well as in primary leukemia patient samples.Mechanistically, R-2HG directly binds to and inhibits the enzymaticactivity of FTO, a major demethylase of N⁶-methyladenosine (m⁶A) that isthe most abundant internal modification of messenger RNA (mRNA)²³ thatresults in increased global m⁶A modification and down-regulation of MYCsignaling. The high abundance of FTO and the hyper-activation of MYCsignaling confer R-2HG sensitivity and resistance, respectively, inleukemic cells. Pharmaceutical inhibition (e.g., by JQ1²⁴) of MYCsignaling sensitizes R-2HG-resistant leukemic cells to R-2HG treatment.R-2HG also exhibits a synergistic or additive effect with standardtherapeutic agents on inhibiting leukemic cell viability. Moreover,R-2HG also displays anti-tumor effect on a variety of human brain tumorcell lines.

EXAMPLES

The Examples are set forth to describe and support embodiments of theinvention by providing detailed illustration of specific aspects andelucidation of underpinning mechanisms. The scope of the inventionshould not be construed as limited to the illustrated embodiments andaspects, but is understood to be commensurate with the appended claims.

The following assays and methodologies apply generally to theExperiments set forth below.

Culture of cell lines and treatment with R-2HG. For leukemia cells,U937, THP1, MV4-11, JURKAT and HEL were obtained from America TypeCulture Collection (ATCC) and cultured at 37° C. in RPMI with 10% fetalbovine serum (FBS) (Gemini Bio-Products), 1% Penicillin-Streptomycin(Life Technologies) and 1% HEPES (Life Technology); TF-1 (ATCC) wasmaintained in RPMI with 10% FBS, 1% Penicillin-Streptomycin, 1% HEPESand 2ng/ml GM-CSF (PeproTech); K562 (ATCC) was cultured in IMDM with 10%FBS, 1% Penicillin-Streptomycin and 1% HEPES; NOMO-1, ML-2, PL21, ME-1and NB4 were obtained from DSMZ and kept in RPMI with 10% FBS, 1%Penicillin-Streptomycin and 1% HEPES; SKNO-1 (DSMZ) was maintained inRPMI with 10% FBS, 1% Penicillin-Streptomycin, 1% HEPES and 10ng/mlGM-CSF; KOPN-1, KOCL69, KOCL48, KOCL50, KOCL45 and KOCL51 weremaintained in RPMI with 10% FBS, 1% Penicillin-Streptomycin and 1%HEPES; MA9.3 (MLL-AF9-transformed human CD34+cord blood cell), MA9.3ITD(MLL-AF9 plus FLT3-ITD), MA9.3RAS (MLL-AF9 plus NRasG12D), MA9.6 (MLL),MA9.6ITD (MLL-AF9 plus FLT3-ITD) and MA9.6RAS (MLL-AF9 plus NRasG12D)were established by the Mulloy group²⁶. For the gliablastoma cell lines,including 8MGBA, A172, U87MG, GAMG, T98G, LN229, LN18 and DK-MG, wereoriginally maintained by the Plas group. All of the cells, withexception of DK-MG, were cultured in DMEM with 10% FBS, 1%Penicillin-Streptomycin and 1% HEPES; DK-MG was maintained in RPMI with10% FBS, 1% Penicillin-Streptomycin and 1% HEPES. All the cells are onlyused for research study, and not among commonly misidentified cellslines, and confirmed to be mycoplasma-free. All the cells were treatedwith cell membrane-permeable version of R-2-Hydroxyglutarate (R-2HG)(Toronto Research Chemicals) with indicated concentration.

Leukemic Patient and Healthy Control Samples and In Vitro Colony FormingAssays.

All the AML patient samples were obtained at the time of diagnosis orrelapse and with informed consent at the University of Chicago Hospital(UCH), City of Hope (COH) or the First Affiliated Hospital of ZhejiangUniversity, and were approved by the institutional review board of theinstitutes/hospitals. The information about AML patients was exhibitedin Table 3. The BM mononuclear cells (MNCs) were isolated with NycoPrep1.077A (Axis-Shield) and stored at liquid nitrogen until used. Thehealthy PB and BM MNCs were purchased from AllCells; the healthy CD34+hematopoietic stem/progenitor cells (HSPCs) and CD34− cells wereisolated from cord blood samples, which were purchased from CincinnatiChildren's Hospital. For colony forming assay of BM progenitors, 10,000cells were plated in 24-well plate with 1 mL human methylcellulosecomplete media (R&D Systems) and the colonies were counted 12 dayslater.

TABLE 3 The detailed information for AML patients. Sample ID Molecularabnormalities Cytogenetics 22162 — t(11; 19) 20507 — t(8; 21) 13295 —t(9; 11) 9084 — t(9; 11) 9003 — t(8; 21) A2233 FLT3 ITD Neg., FLT3 TKDNeg., NPM1 Pos., Normal CEBPA Neg., IDH1 Pos., IDH2 Neg. A2535 FLT3 ITDNeg., FLT3 TKD Neg., NPM1 Neg., 47, XY, C-Kit Neg., CEPBA Neg., IDH1Neg., IDH2 Pos. +8[20] A2786 FLT3 ITD Neg., FLT3 TKD Neg., NPM1 Neg.,Normal CEPBA Neg., IDH1 Neg., IDH2 Pos. A2324 FLT3 ITD Neg., FLT3 TKDNeg., NPM1 Neg., Normal IDH1 Neg., IDH2 Pos. A2061 FLT3 ITD Neg., FLT3TKD Neg., IDH1 Neg., Normal IDH2 Pos. A1951 FLT3 ITD Neg., FLT3 TKDNeg., NPM1 Neg., Trisomy 8 CEPBA Neg., IDH1 Neg., IDH2 Pos., BCR-ABLA2408 FLT3-ITD Neg., FLT3-TKD Neg., CEBPA Neg., Del(8) IDH1 Neg., IDH2Neg., NPM1 Neg., JAK2 Neg. A2418 FLT3 ITD Neg., FLT3 TKD Neg., NPM1Neg., Del (5) C-Kit Neg., CEBPA Neg., IDH1 Neg., IDH2 Neg.

Cell proliferation/viability, cell cycle and cell apoptosis assays. Tostudy the effects of R-2HG, FTO, or IDH1 ^(R132H) on viability, thecells were seeded into 96-well plates at the concentration of5,000-10,000 cells/well in triplicates and MTT (G4000, Promega) was usedto assess cell proliferation and viability following the manufacturer'sinstructions. For cell cycle analysis, Propidium iodide (PI) DNAstaining was used to assess the cells at G0/G1, S and G2/M phases, whileHoechst 33342 and Pyronin Y were used to determine the cells at G0, G1and S/G2/M stages. For the PI staining, cells were resuspended inKrishan's reagent (0.05 mg/ml PI, 0.1% trisodium citrate, 0.02 mg/mlribonuclease A, 0.3% NP-40), incubated at 37° C. for 30 minutes and thenapplied to the flow cytometer.; For Hoechst/Pyronin Y staining, thecells were suspended in cell culture medium, incubated at 37° C. for 45minutes with existence of 10 ug/mL Hoechst 33342 and further incubatedat 37° C. for 15 minutes with existence of Pyronin Y before flowcytometry. Cell apoptosis assay was conducted with FITC Annexin VApoptosis Detection Kit I (BD Pharmingen) according to themanufacturer's instructions.

“human-in-mouse” xeno-transplantation models. The NOD/LtSz-scidIL2RG-SGM3 (NSGS) mice were used for “human in mouse”xeno-transplanation model. The NSGS mouse was created by the Mulloygroup²⁷. NOMO-1 and MA9.3ITD cells, exposed to 300 uM R-2HG or PBS for 4days, were collected, washed twice with PBS and transplanted via tailvein injection into 6- to 8-week-old NSGS recipient mice. For eachrecipient mouse, 0.2-0.5×10⁶ human leukemia cells were transplanted. Themice were euthanized by CO2 inhalation if they displayed typicalleukemic symptoms, i.e. hunched posture, labored breathing and decreasedactivity.

Flow cytometry. All the samples were analyzed by FACSAria II orLSRFortessa cell analyzer (BD Bioscience). Flow cytometry analysis ofmouse BM cells were performed as described previous⁴⁶ with somemodifications. Data were analyzed with FlowJo software. The followingantibodies were used for staining cells, Pacific bluelabeled-anti-mouse/human CD11b (Mac-1) (BioLegend), APC labeledanti-mouse CD117 (c-kit) (2B8) (eBioscience), PE-conjugated anti-humanCD45 (ThermoFisher), FITC-labeled Annexin V (BD Pharmingen), propidiumiodide (PI) (BD Pharmingen), Hoechst 33342 (Sigma) and Pyronin Y(Sigma).

Plasmid construction. The wild type FTO-CDS and mutant FTO-CDS (codingregion sequence) were amplified from pcDNA3.1_FTO and pcDNA3.1_mutFTO(the two plasmids were kindly provided by Dr. Chuan He) by PCR using thefollowing primers: forward 5′-AGAGCTCTAGAACCACCATGGATTACAAAGATGAC-3′ andreverse 5′-CTAAGATTGCGGCCGCCTAGGGTTTTGCTTCCAGAAGC-3′, and thensubsequently cloned into lentivector-based pMIRNA1 (SBI). The shRNAsaganist FTO and YTHDF2 were inserted into pLK0.1 vector. The IDH1^(R132H) (provided by the Sasaki group) was inserted into pTRIPZlentiviral inducible vector.

RNA extraction, cDNA synthesis, qPCR and m⁶A dot blot. RNA was extractedwith miRNeasy Mini Kit (QIAGEN) according to the manufacturer'sguidelines. For cDNA synthesis, 200 ng RNA was used for reversetranscription in 10 ul reaction volume with Qiagen's RT kit followingthe manufacturer's instructions. Then qPCR was performed with 2× SYBRgreen qPCR Master Mix (Thermo Fisher) in the AB 7900HT Fast Real-TimePCR system (Applied Biosystem). GAPDH or ACTIN was used as endogenouscontrol and each reaction was run in triplicate. The m⁶A dot blot wasconducted as previously described with some modifications⁴⁷. RNA sampleswere denatured at 65° C. for 5 minutes with existence of 3 volume of RNAincubation buffer, added equal volume of chilled 20× SSC buffer(Sigma-Aldrich), and spotted on the Amersham Hybond-N+ membrane (GEHealthcare) with a Bio-Dot Apparatus (Bio-Rad). After UV crosslinking,the membrane was washed with 1XPBST buffer (Thermo Scientific), blockedwith 5% non-fat milk and incubated with anti-m⁶A antibody (SynapticSystems) overnight at 4° C. Then the HRP-conjugated goat anti-rabbit IgG(Santa Cruz Biotechnology) was added to the blots for 1 hour at roomtemperature and the membrane was developed with Amersham ECL PrimeWestern Blotting Detection Reagent (GE Healthcare). The relative signaldensity of each dot was quantified by Gel-Pro analyzer software.

DNA extraction and 5 hmC dot blot. DNA was isolated with DNeasy Blood &Tissue Kit (Qiagen) according to the manufacturer's instructions. Toassess 5 hmC levels, dot blot was performed as follows: DNA samples wereadded into 0.1N NaOH, denatured at 99° C. for 5 minutes, neutralized byadding 0.1 volume of 6.6M ammonium acetate, and spotted on AmershamHybond-N+. After UV crosslinking, the membrane was staining with 0.02%methylene blue (Sigma-Aldrich), washed with 1XPBST buffer, blocked with5% non-fat milk and incubated with 5 hmC antibody (Active Motif)overnight at 4° C. Then the HRP-conjugated goat anti-rabbit IgG (SantaCruz Biotechnology) was added to the blots for 1 hour at roomtemperature and the membrane was developed with Amersham ECL PrimeWestern Blotting Detection Reagent (GE Healthcare). The relative signaldensity of each dot was quantified by Gel-Pro analyzer software.

Protein extraction and western blotting. For western blotting, cellswere placed on iced, washed twice with ice-cold PBS. Proteins wereextracted with RIPA buffer (Sigma-Aldrich) with protease inhibitorcocktail and phosphatase inhibitor cocktail (Thermo Fisher). The proteinconcentration was determined with BCA protein assay kit (ThermoScientific). An estimated 30-60 ug protein was loaded per well on 10%SDS-PAGE gel and transferred onto PVDF membrane (Fisher Scientific),activated by methanol. Membranes were washed with 1× PBST, blocked with5% milk and incubated with antibodies against FTO (ab124892, Abcam),ALKBHS (ab174124, Abcam), GAPDH (sc-47724, Santa Cruz), β-Actin (3700S,Cell Signaling), MYC (sc-764, Santa Cruz), Flag (F1804, Sigma Aldrich),H3K9me3 (ab8898, Abcam) and H3K36me3 (ab9050, Abcam). Secondaryantibodies and detection were according to routine laboratory practices.

Drug affinity responsive targets stability (DARTS). To identify thepotential target of R-2HG, DARTS was conducted following the publishedprotocol⁴⁸. 50×10⁶ cells were lysed in M-PER (78501, Thermo FisherScientific) with protease inhibitor cocktail and phosphatase inhibitorcocktail. TNC buffer (50 mM Tris-HCL pH8.0, 50 mM NaCl and 10 mM CaCl₂)was added into the lysate and the protein concentration was determinedby BCA assay. Cell lysates were incubated with varying concentration ofR-2HG or PBS (vehicle) for 1 hour at room temperature and digested withPronase (1:300 for ALKBHS; 1:1000 for FTO) (10165921001, Roche) for 30minutes at room temperature. The digestion was stopped by proteaseinhibitor cocktail and the samples were immediately placed on ice. ForR-2HG target identification, western blot was performed. GAPDH was usedas a negative control.

Lentivirus preparation, precipitation and infection. Lentivirusparticles for pMIRNA1-FTO, pMIRNA1-FTO-Mut, pMIRNA1, pLK0.1-shFTO,pLK0.1-shYTHDF2 and pLK0.1 were packaged with pMD2.G, pMDLg/pRRE andpRSV-Rev (Addgene). Briefly, 0.5 μg pMD2.G, 0.3 μgpMDLg/pRRE, 0.7 μgpRSV-Rev and 1.5 μg construct for overexpression or knockdown ofspecific genes were co-transfected into HEK-293T cells in 60 mm cellculture dish with Effectene Transfection Reagent (301427, QIAGEN). ThepTRIPZ-IDH1^(R132H) was packaged with psPAX2 and pMG2.G. The lentivirusparticles were harvested at 48 and 72 hours and concentrated with PEG-itvirus precipitation solution (LV810A-1, SBI). For infection, thelentivirus were directly added into with cells with existence of 8 ug/mlpolybrene (H9268, Sigma-Aldrich) and then spinoculation was conducted at32° C., 1000 rmp for 90 min. The positive infected cells were selectedwith GFP expression (for FTO and FTO-Mut) or 1 ug/ml puromycin (forshFTO, shYTHDF2 and IDH1 ^(R132H)) (P8833, Sigma-Aldrich). Afterselection, 1 ug/ml Doxycycline (D9891, Sigma-Aldrich) was added to toinduce expression of IDH1 ^(R132H).

RNA-seq and relative data analysis. RNA from R-2HG sensitive, resistantand healthy controls cell lines were extracted by mirVana miRNAIsolation Kit (Thermo Fisher, Grand Island, N.Y.) with total RNAextraction protocol. NEBNext Poly(A) mRNA Magnetic Isolation Module (NewEngland BioLabs, Ipswich, Mass.) was used for polyA RNA purification.Library was prepared by PrepX mRNA Library kit (WaferGen) combinedApollo 324 NGS automated library prep system. Libraries at the finalconcentration of 15 pM were clustered onto a single read (SR) flow cellusing Illumina

TruSeq SR Cluster kit v3, and sequenced to 50 bp using TruSeq SBS kit onIllumina HiSeq system. Differential gene expression was analyzed bystandard Illumina sequence analysis pipeline. The data have beendeposited in the GEO repository with the accession number GSE87187.

RNA samples from R-2HG- or PBS-treated sensitive leukemia cells werealso extracted, purified as described above, library was prepared by orNEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs,Ipswich, Mass.). The libraries were sequenced and analyzed following thesame protocol as above. The data have been deposited in the GEOrepository with the accession number GSE87189.

Gene Set Enrichment Analysis (GSEA)²⁸ was used to analyze the signalpathway enrichment in different groups of samples. “H: hallmark genesets” and “C2: curated gene sets” obtained from The Molecular SignaturesDatabase (MsigDB)²⁸ were used as the “gene sets database” input.

m⁶A-seq assays and data analysis. The m⁶A-seq procedure was performed aspublished protoco1³⁶. Total RNA was isolated with TRIZOL (15596-018,Life technology). Polyadenylated RNA was extracted using FastTrack MAGMaxi mRNA isolation kit (Life technology). RNA fragmentation Reagents(Ambion) was used to randomly fragment RNA. m⁶A antibody (SynapticSystems) was applied for m⁶A pull down (i.e., m⁶A IP). Both input andm⁶A IP samples were prepared for next-generation sequencing (NGS). Thelibrary preparation was constructed by TruSeq Stranded mRNA Sample PrepKit (Illumina) and was quantified by BioAnalyzer High Sensitivity DNAchip, and then was deeply sequenced on the Illumina HiSeq 2500. The datahave been deposited in the GEO repository with the accession numberGSE87190.

For the data analysis, the following pipeline was used to identify m⁶Apeaks. The reads from input and m6A IP samples were aligned to GRCh38reference genome using Tophat⁴⁹. Both MACS2⁵° callpeak function withparameter extsize 85 and exomePeak⁵¹ with default settings were used tocall m⁶A peaks based on the .bam files generated by Tophat. To achievehigh specificity, only the m⁶A peaks called by both MACS2 and exomePeakwere retained for the further analysis. The m⁶A peaks were annotatedusing an ad hoc perl script. Sequence motifs enriched in m⁶A peakregions compared to control regions were identified using HOMER⁵². Thedifferentially methylated m⁶A peaks were also identified by MACS2bdgdiff function and exomePeak, the peaks called by both MACS2 andexomePeak were retained. Circos⁵³ and Integrative Genomics Viewer(IGV)⁵⁴ were used to visualize the distributions of the m6A peaks. TheRNA-seq reads were normalized using Cufflinks⁵⁵. Cuffdiff⁵⁶ was used tocalculate differentially expressed genes.

Gene-specific m⁶A qPCR. To assess the relative abundance of specificmRNA in m⁶A IP and input groups, qPCR was performed. The m⁶A RNAimmunoprecipitation (MeRIP) was performed with Magna MeRIP m⁶A kit(17-10499, Millipore) according to the manufacturer's instructions.Reverse transcription and qPCR were performed with Qiagen's RT kit and2X SYBR green qPCR Master Mix. Cycle threshold (C_(t)) values were usedto determine the relative enrichment of mRNA.

RNA stability assay. The actinomycin D (A9415, Sigma-Aldrich) was addedinto leukemia cells at 5 ug/ml to assess RNA stability. After 0, 2, 3, 4or h hours of incubation, the cells were collected, RNA samples wereextracted for reverse transcription and qPCR. The mRNA degradation ratewas estimated according to the published paper⁵⁷. With actinomycin D,the mRNA transcription was closed and the degradation rate of RNA(K_(decay)) was estimated by following equation:

In(C/C ₀)=−K _(decay) t

C₀ is the concentration of mRNA at time 0 hour. And t is thetranscription inhibition time, C is the mRNA concentration at the timet. Thus the K_(decay) can be derived by the exponential decay fitting ofC/C₀ versus time t. The half-time (t_(1/2)), which meansC/C₀=50%/100%=½, can be calculated by the following equation:

In(½)=−K _(decay) t _(1/2)

Rearrangement of the above equation leads to the mRNA half-life timevalue, t_(1/2)=In2/K_(decay).

Dual-Luciferase reporter and mutagenesis assays. To determine whetherFTO-induced expression of MYC is dependent on m⁶A modification, weperformed dual-luciferase reporter and mutagenesis assays withpMIR-REPORT-MYC-CDS-WT (wild type CDS of MYC), pMIR-REPORT-MYC-CDS-Mut(mutant CDS of MYC, m⁶A was replaced by T in the m⁶A motifs),pGL3-Basic-MYC-5′UTR-WT (wild type 5′UTR of MYC) andpL3-Basic-MYC-5′UTR-Mut (mutant 5′UTR of MYC, m⁶A was replaced by T inthe m6A motifs). All the plasmids were transfected into HEK-293T cellswith pRL-TK (control reporter vector) and pMIRNAl-FTO, orpMIRNAl-FTO-Mut or pMIRNA1.

The relative luciferase activities were assessed with Dual-luciferasereporter assay system (E1910, Promega) at 48 hours. Each group wasrepeated in triplicate.

Example 1 R-2HG Shows a Broad Anti-Leukemic Activity

To define the pathological effect of R-2HG in leukemia in general, 27leukemia cell lines (Table 1) were exposed to a series of concentrations(i.e., 20, 100 and 300 μM) of cell membrane-permeable ester-modifiedR-2HG (as set forth structurally herein).

Very strikingly, R-2HG inhibited cell growth/proliferation and viabilityin a time- and dose-dependent manner in the vast majority of theleukemia cell lines, though with variable inhibitory degrees; nopromoting effect on cell growth/viability was observed (FIG. 1A and FIG.1B; FIG. 6A-6AA, FIG. 7A-7AA). The inhibition of cellproliferation/viability is likely related to R-2HG-induced cell-cyclearrest and apoptosis (FIG. 1C-1E; FIG. 8A-8C). Since it was previouslyreported that R-2HG promoted cell proliferation and induced leukemictransformation in TF-1 cells under a GM-CSF poor condition¹³, it wasconsidered important to attempt to replicate the experiments. Consistentwith the previous report¹³, the data show that R-2HG slightly decreasedcell growth/viability in normal condition (2 ng/ml of GM-CSF), whilesignificantly increasing cell proliferation in cytokine-poor condition(0.1 ng/ml of GM-CSF) (FIGS. 9A and 9B). Nonetheless, in SKNO-1, anotherGM-CSF-dependent leukemia cell line²⁵, R-2HG notably inhibited cellproliferation and viability at cytokine-normal and -poor conditions(FIGS. 9C and 9D), suggesting that the proliferation-promotion effect ofR-HG observed in TF-1 cells is unique and not simply due to a lowconcentration of GM-CSF. Moreover, it was shown that R-2HG alsodecreased colony-forming activity (FIG. 1F) and cell viability (FIG. 1G)of human primary AML cells.

TABLE 1 The selected 27 leukemia cell lines for detecting R-2HGresponse. Cell line name Diagnosis Karyotype HEL AML-M6 Normal JURKATATL Normal K562 CML t(9; 22) KASUMI-1 AML-M2 t(8; 21) KOCL-45 ALL-L1t(4; 11) KOCL-48 AML-M4 t(4; 11) KOCL-50 ALL-L1 t(11; 19) KOCL-51 ALL-L1del(11)(q23) KOCL-69 ALL-L1 t(4; 11) KOPN-1 ALL-L1 t(11; 19) MA9.3 AMLNormal MA9.3ITD AML Normal MA9.3RAS AML Normal MA9.6 AML Normal MA9.6RAS AML Normal MA9.6ITD AML Normal ME-1 AML-M4 inv(16) ML-2 AML-M4 t(6;11) MONOMAC 6 AML-M5 t(9; 11) MV4-11 AML-M5 t(4; 11) NB4 AML-M3 t(15;17) NOMO-1 AML-M5 t(9; 11) PL-21 AML-M3 t(15; 17) SKNO-1 AML-M2 t(8; 21)TF-1 AML-M6 Normal THP-1 AML-M5 t(9; 11) U937 AML-M5 t(10; 11) Note:AML, acute myeloid leukemia; CML, chronic myeloid leukemia; ALL, acutelymphoid leukemia; ATL, adult T-cell leukemia. MA9.3 and MA9.6 arederived from CD34+ HSPCs transformed with MLL-AF9 fusion gene; MA9.3ITDand MA9.6ITD are transformed with while MLL-AF9 plus FLT3-ITD; MA9.3RASand MA9.6RAS are transformed with MLL-AF9 plus NRasG12D.

The “human-in-mouse” xeno-transplantation leukemic model was selected toevaluate the effect of R-2HG on in vivo leukemia progression. Twosensitive cells, NOMO-1 and MA9.3ITD (MLL-AF9 plus FLT3-ITD-transformedhuman cord blood CD34⁺ cells)²⁶, were treated with R-2HG for 4 days invitro and then directly injected into NSGS (NOD-scid IL2Rgnu11-3/GM/SF,NSG-SGM3)²⁷ mice by tail vein injection (FIG. 1h ). Compared with thecontrol group, mice injected with R-2HG-treated cells experienceddelayed development of full-blown AML (FIG. 1I), with suppressedsplenomegaly and hepatomegaly (FIG. 1J), inhibited engraftment ofleukemic cells (FIG. 1K, FIGS. 10A and 10B) and decreased of leukemicimmature blast cells (FIG. 10C and 10D). Thus, the in vitro and in vivoevidence demonstrate the unanticipated broad anti-leukemic activity ofR-2HG, which was previously widely considered as an oncometabolite.

Example 2

Factors Correlating with R-2HG Sensitivity in Leukemic Cells

Functionally, R-2HG acts as a competitive inhibitor ofFe(II)/α-ketoglutarate (α-KG)-dependent dioxygenases (Table 2)⁹. Todetermine which dioxygenase(s) and signaling pathway(s) are responsiblefor the response of leukemic cells to R-2HG, RNA-seq with 4(R-2HG-)sensitive and 5 resistant leukemia cell lines, along with 4healthy control samples were performed (FIG. 2A). A set of dioxygenaseswere identified to be highly expressed in R-2HG-sensitive AML cells andexhibit a positive correlation in expression with the degree of R-2HGinhibitory effect across the AML samples (FIG. 2A, FIG. 11A); thus, theyare potential targets of R-2HG that mediate R-2HG effect. A qPCRanalysis with an expanded cohort of leukemic and normal control samplesconfirmed the positive correlation between gene expression levels anddegrees of R-2HG effect across leukemic samples for 7 out of the top 10dioxygenase genes (FIG. 2A and FIG. 11B-11K); however, only FTO isexpressed at a significantly higher level in leukemic samples comparedto all three types of normal control (mononuclear, CD34⁺ and CD34⁻)cells (FIG. 11J). RNA-seq assays of NOMO-1 and MA9.3ITD samples with andwithout R-2HG treatment were then conducted. It was found that FTO isalso amongst the dioxygenase genes that are significantly down-regulatedby R-2HG in both leukemic cell lines (FIG. 2B and FIG. 12A-12C).

TABLE 3 List of the potential a-KG-dependent/related enzymes. ALKBH1ATP50 JMJD6 LCP1 PLOD1 ALKBH2 BBOX1 JMJD6 LEPRE1 PLOD2 ALKBH3 BBOX2JMJD7 LEPREL1 PLOD3 ALKBH4 DDX5 JMJD8 LEPREL2 SHMT2 ALKBH5 EEF2 KDM2AMINA TET1 ALKBH6 EGLN1 KDM2B NO66 TET2 ALKBH7 EGLN2 KDM3A OGFOD1 TET3ALKBH8 EGLN3 KDM3B OGFOD2 TMLHE APHD1 FIH1 KDM4A P4HA1 UTY APHD2 FTOKDM4B P4HA2 ASPH HIF IAN KDM4C P4HA3 ATP5A1 HR KDM4D P4HB ATP5B HSP90AA1KDM5A P4HTM ATP5C1 HSPA8 KDM5B PAHX-AP1 ATP5D HSPBAP1 KDM5 C PHF2 ATP5EHSPD 1 KDM5D PHF8 ATP5F1 JARID2 KDM6A PHYH ATP5G1 JHDM1C KDM6B PHYHATP5I JMJD1C KDM7A PHYHD1 ATP5J JMJD4 KDM8 PKM2

Through gene set enrichment analysis (GSEA)²⁸ of the two RNA-seqdatasets, 7 gene sets were identified that are strongly correlated withR-2HG sensitivity/response, especially the MYC targets sets (FIG. 2A-2Cand FIG. 13A-13G). Among these pathways, MYC, G2M and E2F signaling arehyper-activated in R-2HG-resistant leukemic samples while moderatelyactivated in R-2HG-sensitive samples, relative to normal control samples(FIG. 2D and FIGS. 13A-13G, 14A and 14B), suggesting theirhyper-activation might be responsible for the resistance to R-2HG.Importantly, R-2HG also suppressed the activities of MYC, G2M and E2Fsignaling pathways in R-2HG sensitive cells (FIG. 2B-2D, FIG. 13A-13G,and FIGS. 14C and 14D). qPCR data also confirmed that R-2HG inhibitedthe major component genes of the MYC signaling in sensitive cells, butnot in resistant cells (FIG. 14E-14F). The fact that MYC, G2M and E2Fsignaling act concordantly to regulate G1/S and G2/M cell cycletransition^(29,30) might be the major mechanism by which R-2HG causescell cycle arrest and apoptosis in sensitive cells (see FIG. 1C-1E, andFIG. 8).

Example 3 R-2HG Targets FTO in Sensitive Leukemic Cells

R-2HG has been shown to inhibit the functions of DNA and histonedemethylases such as TET2, JMJD and KDM, leading to hypermehtylated DNAand histones^(11,31,32). FTO and ALKBHS, two major m⁶A demethylases, arealso Fe(II)/α-KG dependent dioxygenases^(33,34), and thus R-2HG may alsotarget them in leukemic cells. To determine which epigenetic (DNA, RNAor histone) modification is responsible for R-2HG's anti-leukemicactivity, expression levels of the genes encoding DNA demethylases(TET1/2/3), m⁶A demethylases (FTO and ASLKBHS) and histone demethylases(KDM2A, KDM4A and JMJD6) in leukemic samples and healthy control sampleswere first analyzed by qPCR. Notably, FTO is the only gene showing asignificantly positive correlation in expression with R-2HG sensitivityacross the leukemia samples, and is also overexpressed in leukemiasamples relative to normal controls (FIG. 15A-15D). Second, R-2HGtreatment was shown to dramatically increase global RNA m⁶A modificationin the sensitive leukemia cells (FIG. 3A), but not in the resistantcells (FIG. 3B). In contrast,

R-2HG caused a decrease in 5-hydroxymethylcytosine (5 hmC) modificationin resistant cells, but not in sensitive cells (FIG. 15E-15F); noconsistent and significant increase in histone methylation was observedin either sensitive or resistant leukemic cells upon R-2HG treatment(FIG. 15G-15H). Together, the data suggests that FTO and the associatedRNA m⁶A modification are the major mediators of R-2HG's anti-leukemiceffect in sensitive cells.

To determine if FTO is a direct target of R-2HG, a drug affinityresponsive targets stability (DARTS) analysis³⁵ was conducted withprotein lysates in gradient R-2HG treated samples (FIG. 3C). Asexpected, the data suggest that R-2HG binds to FTO directly and protectsits degradation induced by proteinase in a dose-dependent manner; nosuch pattern was observed for ALKBHS (FIG. 3D). Interestingly,consistent with the RNA-seq data (FIG. 2B), our Western blot data alsoindicate that R-2HG treatment can substantially decrease protein levelof FTO, but not ALKBHS, in sensitive leukemic cells, but not inresistant cells (FIG. 3E), though the underlying mechanism warrantsfurther investigation. Thus, R-2HG can not only inhibit FTO activity,but also repress its expression.

Finally, functional studies show that knockdown of FTO endogenousexpression by shRNA in R-2HG-sensitive cells (MA9.3ITD and U927)recapitulated the inhibitory effect of R-2HG on cell growth/viability,associated with increased levels in global m⁶A modification (FIG. 3F-3Ivs. FIG. 1A, 1B, FIG. 3A, FIG. 6A-6AA and FIG. 7A-7AA). Conversely,forced expression of wild-type FTO, but not mutant FTO (carrying twopoint mutations, H231A and D233A, which disrupt the enzymatic activityof FT0)³³, significantly promoted cell proliferation and viability anddeceased global m⁶A levels (FIG. 3G-3I). Moreover, we showed thatknockdown of FTO in R-2HG-sensitive leukemic cells significantly reducedthe sensitivity to R-2HG; conversely, forced expression of FTO inR-2HG-resistant leukemic cells sensitized the cells to R-2HG (FIG. 3J).Collectively, our data indicate that R-2HG treatment causes FTOdown-regulation and loss of function, associated with increased globalRNA m⁶A modification, which likely are response for R-2HG'santi-leukemic effect.

Example 4

The R-2HG

IFTO Axis Regulates MYC Expression

As m⁶A modification is the major epigenetic change induced by R-2HG insensitive leukemic cells (FIG. 3A), transcriptome-wide m⁶A-sequencing(m⁶A-seq; FIG. 4A) and RNA-seq of R-2HG- or PBS-treated NOMO-1 cellswere undertaken. The genomic distributions of the m⁶A peaks and the m⁶Amotifs identified from the two samples (FIG. 16A-16E) are consistentwith those reported previously^(36,37). Notably, consistent with the m⁶Adot blot data (see FIG. 3A), amongst the 1,952 m⁶A peaks detected inboth samples, the vast majority (1,415; 72.1%) of them exhibited asignificant (p<0.01) increase in m⁶A abundance in R-2HG-treated cellsrelative to PBS-treated cells, whereas only 27.9% (547) displayed adecreased pattern (FIGS. 4B and 4C). The transcripts with m⁶A levelincreases were also significantly enriched with target genes of MYC, E2Fand G2M signaling (FIG. 4D) and the majority of the hyper-methylated m⁶Apeaks located in CDS, with a greater ratio (63.6%) than expected bychance (52.8%) (FIGS. 16C, 16F and 16G). Combinational analysis ofm⁶A-seq and RNA-seq data shows that dysregulated m⁶A peaks (increased ordecreased in abundance) have no obvious trend in the overall transcriptlevel change in R-2HG-treated cells relative to control cells (FIG.16H).

MYC targets and E2F targets are the top gene sets repressed by R-2HG insensitive leukemic cells (see FIG. 2B), and they are believed to beregulated directly or indirectly by MYC, a master regulator anduniversal amplifier of global gene regulation^(38,39). Notably, them⁶A-seq data from R-2HG- or PBS-treated cells indicates that the MYCtranscripts are enriched with m⁶A peaks, and R-2HG treatment causes aconsiderable increase in the abundance of the m⁶A peaks, especially inthe 5′UTR and CDS regions (FIG. 4E), which was confirmed bygene-specific m⁶A qPCR assays (FIG. 4f ). As MYC is the top 1 gene whoseexpression is dramatically repressed by R-2HG in both NOMO-1 andMA9-3ITD leukemic cells (FIGS. 14C and 14D), determination of whetherthe suppression of MYC expression is related to R-2HG-mediatedrepression of FTO expression/activity and increase of m⁶A abundance onMYC transcripts was undertaken. First, luciferase reporter andmutagenesis assays were performed with MYC 5′UTR or CDS containingwild-type or mutant m⁶A sites (note: m⁶A was replaced with T in themutants). As expected, forced expression of wild-type FTO, but notmutant FTO, relative to the control, significantly increased luciferaseactivity of the reporter construct carrying wild-type MYC 5′UTR or CDS;such increase was abrogated when the putative m⁶A sites were mutated(FIG. 4G). Thus, the data suggest that FTO can promote expression of MYCand such promotion relies on the m⁶A modification on MYC transcripts.

Next, it was shown that R-2HG remarkably suppressed MYC and FTOexpression in sensitive cells, but not in resistant cells (FIGS. 17A and17B). Consistent with the effect of R-2HG, knockdown of FTO alsosubstantially inhibited MYC expression; conversely, compared with forcedexpression of mutant FTO or empty vector, ectopically expressedwild-type FTO promoted MYC expression (FIG. 17C).

To further elucidate the molecular mechanism by which R-2HG-induced m⁶Amodification increase regulates MYC expression, MYC mRNA stability inleukemia cells was assessed with R-2HG treatment or m⁶A readerinhibition. Remarkably, R-2HG dramatically decreased MYC mRNA stabilityin the sensitive cells (half life: 0.72 hours vs. 1.57 hours), whereaswith little effect in resistant cells (FIG. 4H), indicating thatR-2HG-induced down-regulation of MYC is likely related to the lessstability of MYC transcripts due to increased m⁶A modification. Theknockdown of expression of YTHDF2, a major m⁶A reader that isresponsible for the decay of m⁶A-modified mRNA transcripts³⁷ and bindson MYC mRNA (FIG. 16I), noticeably increased stability of MYCtranscripts in leukemia cells (FIG. 4I and FIG. 17D), suggesting thatthe stability of MYC transcripts is at least in part subjected toYTHDF2-mediated RNA decay.

Lastly, m⁶A-seq of R-2HG- and PBS-treated sensitive cells (MA9.3ITD) orresistant cells (MA9.3RAS) with or without FTO knockdown (for sensitivecells) or FTO overexpression (for resistant cells) was performed tofurther analyze the effects of R-2HG and FTO on m⁶A modification of MYCtranscripts. In sensitive cells, R-2HG substantially increased m⁶Aabundance at the 5′UTR and CDS regions of MYC transcripts, and suchincrease can be sufficiently abrogated by FTO knockdown; in contrast, inresistant cells, R-2HG showed no obvious effect on m⁶A abundance on MYCtranscripts, whereas forced expression of FTO substantially reduced them⁶A abundance in PBS-treated cells and such decrease was abrogated byR-2HG treatment, resulting in the substantial increase in m⁶A abundanceat the 5′UTR and CDS regions of MYC transcripts in R-2HG treatedFTO-overexpressing cells than in PBS-treated ones (FIG. 4J).Collectively, the data uncovers a novel molecular mechanism (i.e.,R-2HG-IFTO

m⁶A modification

IMYC) by which R-2HG inhibits MYC signaling and exerts anti-leukemiceffect (FIG. 4K).

Example 5

FTO/MYC Homeostasis Controls R-2HG Sensitivity and Pre-Treatment withMYC-Signaling Inhibitors Sensitizes Leukemic Cells to R-2HG

Endogenous R-2HG is converted from α-KG by mutant IDH^(7,8). Todetermine whether mutant IDH can recapitulate the phenotypes observed inR-2HG treated leukemia cells, leukemic cell lines with inducibleexpression of mutant IDH were created. As expected, doxycycline-inducedIDH 1 ^(R132H) expression sufficiently mimicked the phenotypes caused byexogenous R-2HG, such as suppression of FTO and MYC expression (FIGS. 5Aand 5B), enhanced m⁶A modification (FIG. 5C), cell cycle arrest (FIG.5D), decreased cell proliferation/growth (FIG. 5E) and increased cellapoptosis (FIG. 5F) in sensitive leukemic cells (NOMO-1 and U937), butnot in resistant cells (NB4).

To address the question of why IDH mutations exist in 10-20% of AMLcases^(17,18), an integrative analysis of the TCGA AML microarraydataset (including 37 IDH-mutant and 160 IDH-wildtype AML patients)⁴⁰with the RNA-seq data shown in FIG. 2A was conducted. Five coresignaling pathways enriched in both IDH-mutant AML samples wereidentified (relative to IDH-wildtype AML samples, in the whole set or inthe normal-karyotype subset) and R-2HG-resistant (relative toR-HG-sensitive) leukemic cells (FIG. 5G and FIGS. 18A-18C). Except forcholesterol homeostasis, four pathways (i.e., MYC targets V1, MYCtargets V2, G2M checkpoint and E2F targets) were also enriched insensitive cells compared with healthy controls (FIG. 18D). IDH-mutantAML samples were confirmed to have a higher expression level of MYC andits critical targets, whereas a lower level of FTO (but not ALKBH5)expression, than IDH-wildtype AML samples (FIG. 5H). The Western blotassays also confirmed that R-2HG-resistant leukemic cell lines have amuch higher level of MYC while a lower level of FTO than the sensitivecell lines; R-2HG treatment caused a substantial decrease in FTO and MYCexpression in the sensitive cell lines, but had only minor effect ontheir expression in the resistant cell lines (FIG. 5I). Thus, it waspresumed that the highly activated MYC signaling diminishes theanti-leukemic effect of R-2HG in IDH-mutant AML cells. To test this, MYCin R-2HG-sensitive leukemic cells were over-expressed and it was foundthat forced expression of MYC did in fact render the leukemic cellsresistant to R-2HG treatment (FIG. 5J). Conversely, pharmaceuticalinhibition of MYC signaling by JQ1²⁴ notably increased sensitivity ofR-2HG-resistant leukemic cells to exogenous R-2HG or IDH1 ^(R132H), andcombination of JQ1 and R-2HG exhibited a stronger inhibition on cellviability (FIG. 5K). Primary AML cells with IDH mutations are alsosensitive to JQ1 treatment, often with an IC₅₀ lower than 1 μM, thoughwith a higher IC₅₀ than do AML cells with wild-type IDH (FIG. 18E),likely due to the hyper-activation of MYC signaling in the former. Takentogether, the data suggest that high abundance of FTO confers R-2HGsensitivity in leukemic cells, whereas hyper-activation of MYC rendersleukemic cells resistant to R-2HG. Exemplary MYC pathway inhibitors areset forth in Table 4.

TABLE 4 Small molecules known to inhibit MYC signaling Compound nameClass Target References Flavopiridol CDK inhibitor Cdk-9 Chen et al.2005; Rahl et al. 2010 Purvalanol A CDK inhibitor Cdk-1 Goga et al. 2007SU9516 CDK inhibitor Cdk-2, Cdk-9 Gao et al. 2006 PHA 767491 CDKinhibitor Cdc-7 and Montagnoli et al. HCI Cdk-9 2008; Natoni SNS-G32 CDKinhibitor Gdfc-2, Cdk-7, et al. 2011 and Cdk-9 Walsby et al. 2011 JQ1BET Brd-4, Brd-3, Filippakopoulos bromodomain et al. 2010; inhibitorBrd-2 Delmore et al. 2011 SGI-1776 PIM kinase Pim-1 Zippo et al.inhibitor 2007, 2009 EPZ004777 Dot1 L inhibitor Pim-1 Daigle et al. 2011C464 p300/CBP p300 McMahon et ACTfrase al. 1998 inhibitor SAHA HDACinhibitor Triptolide TFIIH/XPB XPB Titov et al. 2011 Nutlin-3a p53-MDM2p53-MDM-2 Felsher et al. 2000

Example 6 R-2HG Sensitizes Leukemia and Glioma to Chemotherapies

Interestingly, R-2HG also exhibits a synergistic, or at least additive,effect with a series of first-line chemotherapy drugs such as all-transretinoic acid (ATRA), Azacitidine (AZA), Decitabine, and Daunorubicin(FIG. 18F), though the underlying molecular mechanisms need to be forfully delineated. Thus, R-2HG can be readily administered in combinationwith such therapeutic agents to treat leukemia in clinic.

IDH mutations also occur in >70% of patients with lower-grade (II-III)brain tumors and <10% of glioblastomas (grade IV), and such mutationsare usually associated with favorable overall survival^(1,2,19). Inanalysis of the pathological effect of R-2HG in 8 human brain tumor celllines, R-2HG significantly inhibited the proliferation and viability ofall the tumor cells (FIGS. 19A-19C). Thus, these data indicate thatR-2HG also exhibits anti-tumor activity in brain tumors, which maycontribute to the favorable survival of patients with IDH mutations.

The following references are cited herein to support certain backgroundstatements and/or to provide detailed disclosure of known methodologiesor mechanistic models and presence on this list should not be construedas an admission of relevance to patentability.

The entire disclosures of all references on this list are incorporatedherein by citation.

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1. A method of treating a tumor in a subject in need thereof comprising administering to the subject an effective amount of R-2-hydroxyglutarate (R-2HG).
 2. The method of claim 1, wherein the tumor is a brain tumor.
 3. The method of claim 2, wherein the tumor is a glioma.
 4. A method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of R-2HG.
 5. The method of claim 4, wherein the cancer is selected from the group consisting of leukemia and brain cancer.
 6. The method according to claim 1, further comprising administering at least one agent effective for inhibiting MYC signaling prior to administering the R-2HG.
 7. The method according to claim 6, wherein the at least one agent effective for inhibiting MYC signaling is selected from the agents set forth in Table
 4. 8. The method according to claim 6, wherein the patient is characterized by possessing a mutant form of IDH1 and/or an IDH2.
 9. The method according to claim 1, further comprising administering one or more chemotherapeutic agents in conjunction with the R-2HG.
 10. The method according to claim 9, wherein the one or more chemotherapeutic agents are selected from all trans retinoic acid (ATRA), azacitidine (AZA), daunorubicin, and decitabine.
 11. The method according to claim 1, wherein the R-2HG comprises ester-modified R-2HG. 12-20. (canceled) 